N-Hydroxysuccinimide (NHS) activated ZW800-1A and ZW800-1C were conjugated to Cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide (cRGDyK; MW 619.6, AnaSpec), bovine serum albumin (BSA; MW 67 kDa, Sigma-Aldrich), mouse IgG (IgG; MW 150 kDa, Sigma-Aldrich), and poly-ε-lysine (MW 4 kDa, Chisso Corp, Yokohama, Japan), respectively. cRGD-ZW800 conjugates were separated by preparative HPLC system (Waters, Milford, MA, USA) equipped with a PrepLC 150 mL fluid handling unit, a manual injector (Rheodyne 7725i), a 2487 dual wavelength absorbance detector (Waters) and an evaporative light scatter detection (ELSD, Richards Scientific, Novato, CA, USA). BSA-, IgG-, and poly-ε-lysine-ZW800 conjugates were purified by gel-filtration chromatography (GFC) using Bio-Scale Mini Bio-Gel P-6 Desalting columns (Bio-Rad). The labeling ratio was calculated from the ratio of extinction coefficients between the BSA, antibody, and ZW800 fluorophores. See Figure S5 in Supplementary Information for detailed analyses.
Preparative hplc system
Manufactured by Waters Corporation
Sourced in United States
The Preparative HPLC system is a high-performance liquid chromatography (HPLC) instrument designed for the purification and separation of large-scale quantities of chemical compounds or biomolecules. It employs a high-pressure liquid flow to efficiently separate and isolate target analytes from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
2487 dual wavelength absorbance detector, by Waters Corporation (2 mentions)
1525 pump, by Waters Corporation (2 mentions)
2707 autosampler, by Waters Corporation (2 mentions)
2998 pda detector, by Waters Corporation (2 mentions)
6130 quadrupole lc ms, by Agilent Technologies (2 mentions)
Xbridge c18 column, by Waters Corporation (2 mentions)
1200 series hplc, by Agilent Technologies (2 mentions)
Microtof, by Bruker (2 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
P 1010 polarimeter, by Jasco (1 mentions)
Ft ir spectrometer paragon 1000, by PerkinElmer (1 mentions)
Uvmini 1240, by Shimadzu (1 mentions)
Nucleosil c18, by Macherey-Nagel (1 mentions)
Microflex instrument, by Bruker (1 mentions)
Symphony x, by Protein Technologies (1 mentions)
Silica gel, by Merck Group (1 mentions)
Deae cellulose, by Merck Group (1 mentions)
0.5 m hcl, by Merck Group (1 mentions)
Xk26 20 column, by GE Healthcare (1 mentions)
18 protocols using preparative hplc system
1
Conjugation of ZW800 Fluorophores
2
Spectroscopic Characterization of Compounds
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NMR spectra were recorded in deuterated methanol (CD3OD) on a Bruker 600 MHz and were referenced using the residual 1H signal of deuterated solvent at 3.30 ppm [49 (link), 53 (link)]. FT-IR was measured on Perkin–Elmer FT-IR Spectrometer Paragon 1000. UV measurements were performed using a Shimadzu UV mini-1240. Optical rotations were measured on a JASCO P-1010 polarimeter. HPLC purification was performed on Waters preparative HPLC system comprised of 1525 pump, 2707 autosampler, 2998 PDA detector, and Water fraction collector III. The columns used were SunFire C18 column (5 µm, 100 Å, 10 × 250 mm) and SunFire C18 column (10 µm, 100 Å, 19 × 150 mm). TLC was performed using aluminum plates coated with silica gel 60 F254 (Merck). All organic solvents and chemicals were of analytical or LCMS grade, depending on the experiment.
3
Synthesis and Purification of Modified Human NPY Peptide
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Human NPY peptide (YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2) terminated at the N-terminus with 5-hexynoic acid was prepared
using Fmoc chemistry protocols on a multichannel peptide synthesizer.25 (link) Sidechain deprotection and cleavage of the peptides
from the solid support were performed by treatment with reagent K
(88% TFA v/v, 2% triisopropylsilane v/v, 5% dithiothreitol w/v, and
5% water v/v) for 150 min at 20 °C. The peptide was purified
by reversed-phase HPLC (RP-HPLC) using a preparative HPLC system (Waters)
on a Nucleosil C18 (1 × 30 cm) column (Macherey Nagel). The elution
was achieved with a linear gradient of aqueous 0.1% TFA (A) and 0.08%
TFA in acetonitrile (B) at a flow rate of 6 mL/min with UV detection
at 230 nm. The purity of the peptide was controlled by analytical
RP-HPLC on a Waters instrument (Waters Alliance) with a Nucleosil
C18 5 μm column (150 × 4.6 mm) using a linear gradient
of 0.1% TFA in water and acetonitrile containing 0.08% TFA at a flow
rate of 1.2 mL/min. The integrity of the peptide was assessed by LC/MS
using a Thermo Finigan LCQ.
using Fmoc chemistry protocols on a multichannel peptide synthesizer.25 (link) Sidechain deprotection and cleavage of the peptides
from the solid support were performed by treatment with reagent K
(88% TFA v/v, 2% triisopropylsilane v/v, 5% dithiothreitol w/v, and
5% water v/v) for 150 min at 20 °C. The peptide was purified
by reversed-phase HPLC (RP-HPLC) using a preparative HPLC system (Waters)
on a Nucleosil C18 (1 × 30 cm) column (Macherey Nagel). The elution
was achieved with a linear gradient of aqueous 0.1% TFA (A) and 0.08%
TFA in acetonitrile (B) at a flow rate of 6 mL/min with UV detection
at 230 nm. The purity of the peptide was controlled by analytical
RP-HPLC on a Waters instrument (Waters Alliance) with a Nucleosil
C18 5 μm column (150 × 4.6 mm) using a linear gradient
of 0.1% TFA in water and acetonitrile containing 0.08% TFA at a flow
rate of 1.2 mL/min. The integrity of the peptide was assessed by LC/MS
using a Thermo Finigan LCQ.
4
Fmoc-based Peptide Synthesis and Purification
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Peptide syntheses were performed using optimized Fmoc chemistry protocols with a multichannel peptide synthesizer [52 (link)]. Side-chain deprotection and cleavage of the peptide from the solid support were performed by treatment with reagent K (88 % TFA v/v, 2 % triisopropylsilane v/v, 5 % dithiothreitol w/v, 5 % water v/v) for 150 min at 20 °C. The VEGF receptor binding peptides (VRbp) were purified by reversed-phase HPLC (RP-HPLC) using a preparative HPLC system (Waters) on a Nucleosil C18 (1 × 30 cm) column (Macherey Nagel). The elution was achieved with a linear gradient of aqueous 0.1 % TFA (A) and 0.08 % TFA in acetonitrile (B) at a flow rate of 6 mL min−1 with UV detection at 230 nm. The purity of the VEGFR-binding peptide was controlled by analytical RP-HPLC (Fig. S7 ) and their molecular weight was assessed by LC/MS (Fig. S8 ). MS (ESI, m/z): 1967.7 [M+H]+ for CGGGGGGHRHTKQRHTALH and 1967.0 [M+H]+ for HRHTKQRHTALHGGGGGGC.
5
Synthesis and Characterization of IRGB10 Peptides
6
Isolation and Characterization of Galtamycin D
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Streptomyces sp. MBT84 was grown on MM agar plates supplemented with 50 µM catechol, 1% glycerol and 0.5% mannitol at 30 °C for five days. Agar plates were cut into small pieces and soaked in EtOAc to extract metabolites as described earlier. The solvent was subsequently evaporated under reduced pressure at 40 °C to obtain 1.6 g crude extract. This extract was adsorbed onto 1.6 g silica gel (pore size 60 Å, 70–230 mesh, Sigma Aldrich), and loaded on a silica column, followed by gradient elution using mixtures of n-hexane, EtOAc, and MeOH. One of the fractions that eluted with 50% EtOAc: 50% n-hexane was combined with the fraction that eluted with 75% EtOAc: 25% n-hexane, and reconstituted in acetonitrile. This fraction was further purified using a Waters preparative HPLC system comprised of 1525 pump, 2707 autosampler, and 2998 PDA detector. The pooled fraction was injected into a SunFire C18 column (10 µm, 100 Å, 19 × 150 mm). The column was run at a flow rate of 12.0 mL/min, using solvent A (dH2O) and solvent B (acetonitrile), and a gradient of 70-100% B over 20 min to yield 7 (3.4 mg) and 8 (1.9 mg).
Galtamycin D (8): yellow amorphous powder; UV (LC-MS) λmax 222, 266, and 441 nm; HRESIMS m/z 579.1866 [M + H]+ (calcd for C31H31O11, 579.1861); 1H and 13C NMR data (Table S6 ).
7
Purification of Organic Compounds
8
DEAE-Cellulose Column Purification Protocol
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DEAE-cellulose (10 g; Sigma Aldrich, Germany) dry gel washed with distilled water and left at 4°C for 16 h to remove small particles. The swollen gel was suspended in 0.5 M HCl (Merck Millipore, Germany) for 30 min and was filtered and washed with distilled water.The gel was suspended in 0.5 M NaOH (Merck Millipore, Germany) for 30 min (20 ) and washed with phosphate buffer (pH 6.3) 5×. The gel was packed into a XK 26/20 column (GE Healthcare, Sweden) with a 300 cm.h-1 linear velocity (26.5 mL.min-1) using preparative HPLC system (Waters, USA). The packed column was 85 mm bed height and 45 mL bed volume. The column was equilibrated by 3 column volume (CV), 0.07 M buffer phosphate pH 6.3 (19 ,21 (link)) at 225 cm.h-1 linear velocity (20 mL.min-1). Sample was loaded into the column at 150 cm.h-1. Linear flow rate of 13.3 mL.min-1 was applied to separate the adsorbed impurities.
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DEAE-cellulose (10 g; Sigma Aldrich, Germany) dry gel washed with distilled water and left at 4°C for 16 h to remove small particles. The swollen gel was suspended in 0.5 M HCl (Merck Millipore, Germany) for 30 min and was filtered and washed with distilled water.The gel was suspended in 0.5 M NaOH (Merck Millipore, Germany) for 30 min (20 ) and washed with phosphate buffer (pH 6.3) 5×. The gel was packed into a XK 26/20 column (GE Healthcare, Sweden) with a 300 cm.h-1 linear velocity (26.5 mL.min-1) using preparative HPLC system (Waters, USA). The packed column was 85 mm bed height and 45 mL bed volume. The column was equilibrated by 3 column volume (CV), 0.07 M buffer phosphate pH 6.3 (19 ,21 (link)) at 225 cm.h-1 linear velocity (20 mL.min-1). Sample was loaded into the column at 150 cm.h-1. Linear flow rate of 13.3 mL.min-1 was applied to separate the adsorbed impurities.
9
General Synthetic Procedures for Organic Compounds
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All chemicals, unless otherwise
stated, were commercially available and used without further purification.
Reactions were magnetically stirred; commercially available anhydrous
solvents were used. Flash column chromatography (FCC) was performed
using a Teledyne Isco Combiflash Rf or Rf200i; prepacked RediSep Rf
normal phase disposable columns were used. NMR spectra were recorded
on a Bruker Ascend 400. Chemical shifts are quoted in ppm and referenced
to the residual solvent signals: 1H δ = 7.26 ppm
(CDCl3), 4.79 ppm (D2O), 2.50 ppm (DMSO-d6); 13C δ = 77.2 ppm (CDCl3), 39.5 ppm (DMSO-d6); signal
splitting patterns are described as singlet (s), doublet (d), doublet
of doublets (dd), triplet (t), quartet (q), multiplet (m), and broad
(br). Coupling constants (JH–H)
are measured in Hz. High-resolution mass spectra (HRMS) were recorded
on a Bruker microTOF. Low-resolution MS and analytical HPLC traces
were recorded on an Agilent Technologies 1200 series HPLC connected
to an Agilent Technologies 6130 quadrupole LC–MS, connected
to an Agilent diode array detector. Preparative HPLC was performed
on a Gilson preparative HPLC system with a Waters X-Bridge C18 column
(100 mm × 19 mm; 5 μm particle size). Elution conditions
are reported in the general methods. The purity of all compounds was
analyzed by HPLC–MS (ESI) and was >95%.
stated, were commercially available and used without further purification.
Reactions were magnetically stirred; commercially available anhydrous
solvents were used. Flash column chromatography (FCC) was performed
using a Teledyne Isco Combiflash Rf or Rf200i; prepacked RediSep Rf
normal phase disposable columns were used. NMR spectra were recorded
on a Bruker Ascend 400. Chemical shifts are quoted in ppm and referenced
to the residual solvent signals: 1H δ = 7.26 ppm
(CDCl3), 4.79 ppm (D2O), 2.50 ppm (DMSO-d6); 13C δ = 77.2 ppm (CDCl3), 39.5 ppm (DMSO-d6); signal
splitting patterns are described as singlet (s), doublet (d), doublet
of doublets (dd), triplet (t), quartet (q), multiplet (m), and broad
(br). Coupling constants (JH–H)
are measured in Hz. High-resolution mass spectra (HRMS) were recorded
on a Bruker microTOF. Low-resolution MS and analytical HPLC traces
were recorded on an Agilent Technologies 1200 series HPLC connected
to an Agilent Technologies 6130 quadrupole LC–MS, connected
to an Agilent diode array detector. Preparative HPLC was performed
on a Gilson preparative HPLC system with a Waters X-Bridge C18 column
(100 mm × 19 mm; 5 μm particle size). Elution conditions
are reported in the general methods. The purity of all compounds was
analyzed by HPLC–MS (ESI) and was >95%.
10
Comprehensive Analytical Workflow for Metabolite Purification and Characterization
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HPLC purifications were performed
on a Waters preparative HPLC system equipped with a photodiode array
detector (PDA). The absorption was monitored at 220, 290, and 350
nm. LC-MS analysis was performed on a Shimadzu LC-MS 9030 system composed
of a UPLC with an attached PDA, coupled to a QTOF HRMS, which uses
ESI as an ionization source. NMR spectra were acquired on a Bruker
AVIII-600 NMR spectrometer (Bruker BioSpin GmbH). For details on metabolite
extraction and analysis, see theSupplemental Methods .
on a Waters preparative HPLC system equipped with a photodiode array
detector (PDA). The absorption was monitored at 220, 290, and 350
nm. LC-MS analysis was performed on a Shimadzu LC-MS 9030 system composed
of a UPLC with an attached PDA, coupled to a QTOF HRMS, which uses
ESI as an ionization source. NMR spectra were acquired on a Bruker
AVIII-600 NMR spectrometer (Bruker BioSpin GmbH). For details on metabolite
extraction and analysis, see the
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